New Triterpenes from Gymnema sylvestre
by Armando Zarrellia), Marina DellaGreca*a), Afef Ladharib), Rabiaa Haoualac), and
Lucio Previteraa)
a) Department of Chemical Sciences, University Federico II, Complesso Universitario Monte S. Angelo,
Via Cintia 4, IT-80126 Napoli (phone:þ 39-081-674472; fax: þ 39-081-674393; e-mail: dellagre@unina.it)
b) Department of Biology, Faculty of Science of Bizerte, Jarzouna 7021, Tunisia
c) Department of Biological Sciences and Plant Protections, Higher Institute of Agronomy of Chott
Meriem, University of Sousse, 4042, Tunisia (UR03AGR04)
Phytochemical investigation of the aerial parts of Gymnema sylvestre has led to the isolation of seven new triterpenes, six oleane types (5, 7 – 11) and a new lupane type (12), and of the six known analogues 1 – 4, 6, and 13. The structures and relative configurations of these compounds were elucidated by spectroscopic analyses, including 1D- and 2D-NMR spectroscopy and mass spectrometry, and by the comparison of their NMR data with those of related compounds.
Introduction. – Gymnema sylvestre is a bushy climber of the Asclepiadaceae family that is found mainly in the Central and Tropical Indian Peninsula, Sri Lanka, and Africa. It is considered a medicinal plant and is used in folk medicine, ayurveda, and homeopathy for treatment of asthma, eye disorders, inflammation, and snake bites, as a tonic and refrigerant, diuretic, laxative, sedative for coughs, and especially as an antidote for diabetes [1]. Recent studies, based on the historical evidence of the many uses in the medicinal field, have indicated that the leaves and roots of the plant are the parts that contain the biologically active ingredients [2]. The active compounds of G. sylvestre are called gymnemic acids [3]. Their use in the past to counteract the effects of excess glucose in diabetes mellitus explains the Hindi name of the plant, Gur-ma which means destroyer of sugar. The plant extracts have been reported to exert an antimicrobial and hepatoprotective activity, and to lower the levels of triglycerides and LDL cholesterol, and raise the HDL cholesterol (good cholesterol) [4] [5]. They also act as a deterrent against Prodenia eridania, prevent dental caries caused by Streptococcus mutans, and are used in skin cosmetics [6 – 8].
In an investigation of G. sylvestre, we have isolated five known oleanane-type triterpenes, 1 – 4 and 6, six new analogs, 5 and 7 – 11, and two lupane-type triterpenes, 12 and 13, the former of which was new.
Results and Discussion. – The CH2Cl2extract of the aerial parts of G. sylvestre was separated into acidic and neutral fractions with 2n NaOH. The neutral fraction, washed with H2O and concentrated under reduced pressure, was filtered through SiO2 with petroleum ether, CH2Cl2, AcOEt, Me2CO, MeOH, and H2O, as eluents of increasing polarity. Column chromatography of the fraction eluted with CH2Cl2 yielded
compounds 1 – 13. The known triterpenes 1 – 4, 6, and 13 were identified as 3b,16b,28-trihydroxyolean-12-ene (1) [9], 3b,16b,21b,28-tetrahydroxyolean-12-ene (2) [10], 3b,16b,22a,28-tetrahydroxyolean-12-ene (3) [11], 3b,23,28-trihydroxyolean-12-ene (4) [12], 3b,16b,21b,23,28-pentahydroxyolean-12-ene (6) [13], and 3b,16b,29-trihy-droxylup-20(30)-ene (13) [14].
The new compounds were characterized by various spectroscopic methods. The ESI-MS of compound 5 showed a quasi-molecular-ion peak at m/z 475.2 ([Mþ H]þ), suggesting the molecular formula C30H50O4and six degrees of unsaturation. Fragment ions at m/z 457.5 ([M H2Oþ H]þ), 439.3 ([M 2 H2Oþ H]þ), and retro-DielsAlder fragmentation ion, characteristic of an oleanane skeleton, at m/z 250.4 (50%) evidenced an olean-12-ene derivative. The1H- and13C-NMR spectra of 5 confirmed this assumption. The1H-NMR spectrum showed signals of five H-atoms geminal to an O-bearing function as three double doublets at d(H) 4.10, 3.61, 3.52, a doublet at 3.53, and a signal at d(H) 3.31, partly obscured by solvent. The13C-NMR spectrum (Table) exhibited signals of three bearing CH groups at d(C) 74.5, 74.3, 67.8 and of a O-bearing CH2group at d(C) 67.8. In the upfield region of1H-NMR, seven Me singlets were detected at d(H) 1.23, 1.02, 1.01, 0.95, 0.87, 0.81, and 0.71. The analysis of COSY, NOESY, HSQC, and HMBC spectra allowed us to determine the structure of 3b,16b,21b,23-tetrahydroxyolean-12-ene for this compound. The1H,1H-COSY experi-ment showed correlations between HC(16) (d(H) 4.10) and CH2(15) (d(H) 1.67 and 1.25); HC(21) (d(H) 3.52) and CH2(22) (d(H) 1.68 and 1.60); and HC(3) (d(H)
3.61) and CH2(2) (d(H) 1.96 and 1.93). The latter correlated with the CH2(1) (d(H) 1.65, 1.16). Finally, the signal of the olefinic H-atom HC(12) at d(H) 5.28 correlated with HC(11) (d(H) 1.96 and 1.93). The correlations in the HMBC experiment between the H-atom signal at d(H) 4.10 and that of the H-atoms of Me(28) at d(H) 0.81 with the CH signal at d(C) 50.5, assigned to C(18) and bonded to H-atom with the signal at d(H) 2.20, and between the signal of HC(18) with that of the O-bearing HC(16) at d(C) 67.8, indicated an OH group at C(16). The correlations between the signals of HC(23) at d(H) 3.53 and 3.31 with that of the CH at d(C) 48.5, assigned to C(5) and bonded to the H-atom with the signal at d(H) 1.62, and between this signal and that of the O-bearing CH2 at d(H) 67.8 localized another OH group at C(23). Finally, the correlations of the signals of two geminal Me groups, i.e., Me(29) at d(H) 0.95 and Me(30) at d(H) 0.87, with the C-atom signal at d(C) 74.5 defined a further hydroxylation site at C(21). These data, compared with those of the other isolated triterpenes, were in accordance with a 3,16,21,23-tetrahydroxyoleanane skeleton. The
Table. 13C-NMR Data of Triterpenes 5, 7 – 12 (at 125 MHz, in CD
3OD; d in ppm) C-Atom 5 7 8 9 10 11 12 C(1) 40.1 40.1 40.2 39.4 39.1 39.3 40.2 C(2) 25.1 27.9 28.1 37.1 37.5 37.1 28.1 C(3) 74.3 74.3 74.4 219.2 220.1 218.8 74.2 C(4) 43.8 43.7 42.6 51.6 53.9 54.0 43.9 C(5) 48.5 49.3 49.0 (obs)a) 48.7 47.2 47.3 49.4 C(6) 19.6 19.6 19.7 21.0 20.9 21.0 19.5 C(7) 33.9 34.0 36.3 33.5 33.4 33.4 35.4 C(8) 43.8 41.3 43.9 38.0 41.4 41.8 43.9 C(9) 49.0 48.7 52.1 47.4 48.6 48.5 51.9 C(10) 37.7 37.6 38.7 40.0 41.4 37.7 38.6 C(11) 25.1 25.1 23.9 25.3 25.2 25.3 22.4 C(12) 124.5 124.7 27.0 124.4 124.8 125.6 26.7 C(13) 142.3 144.5 139.9 145.2 143.7 142.2 38.5 C(14) 45.2 45.3 48.0 42.2 45.2 44.2 46.2 C(15) 37.0 36.6 37.3 37.1 36.9 36.5 39.0 C(16) 67.8 71.7 78.2 68.3 68.9 69.8 80.0 C(17) 40.1 45.3 45.1 41.8 45.2 47.5 46.2 C(18) 50.5 44.6 129.8 45.6 44.6 43.3 49.8 C(19) 41.6 43.8 40.3 48.1 48.6 47.8 49.6 C(20) 38.2 36.5 34.2 32.2 37.7 37.8 151.6 C(21) 74.5 76.2 36.0 35.3 74.4 78.7 31.5 C(22) 28.8 33.1 30.7 26.6 34.2 74.4 33.9 C(23) 67.8 67.8 67.9 68.5 68.3 69.4 67.7 C(24) 13.2 13.2 13.1 18.5 18.3 18.5 13.0 C(25) 17.0 17.0 17.8 16.4 16.3 16.3 17.5 C(26) 18.1 18.0 19.0 17.8 17.7 17.8 17.7 C(27) 28.1 27.3 23.2 27.9 27.7 28.1 16.8 C(28) 22.9 68.1 64.5 69.5 68.4 59.6 62.4 C(29) 30.3 28.2 32.9 31.8 30.1 30.6 111.1 C(30) 18.3 26.0 25.8 24.8 18.0 19.2 19.9
configurations of the OH groups were deduced from a NOESY experiment (Fig.), where HC(16) showed NOE effects with the H-atoms of Me(27) and H-atom HC(19) (d(H) 2.10), indicating a b-orientation for the OH group at C(16). The HC(21) showed NOE effects with the H-atoms of Me(29), HC(19), and HC(16) indicating also a b-orientation for the OH group at C(21).
The ESI-MS of compound 7 showed a quasi-molecular-ion peak at m/z 491.4 ([Mþ H]þ), suggesting the molecular formula C
30H50O5 and six degrees of unsaturation. Fragment-ion peaks at m/z 473.4 ([M H2Oþ H]þ), 455.3 ([M 2 H2Oþ H]þ), and retro-DielsAlder fragmentation-ion peak, characteristic of an oleanane skeleton, at m/ z 267.5 (30%) evidenced an olean-12-ene derivative. The1H- and13C-NMR data were similar to those of triterpene 6, whose structure had been previously reported in [13]. A concise analysis of its spectroscopic data allowed us to determine the structure of 7 as the C(21)-epimer of the reference compound 6. The1H,1H-COSY experiment showed correlations between: the double doublet HC(16) at d(H) 4.57 and the CH2(15) at d(H) 1.80 and 1.30, the broad triplet HC(21) at d(H) 3.52 and the CH2(22) at d(H) 2.14 and 1.77, abd the double doublet HC(3) at d(H) 3.62 and the CH2(2) at d(H) 1.71. The correlations in the HMBC experiment between the two geminal Me(29) and Me(30) at d(H) 0.92 and 0.95, respectively, with C-atom at d(C) 76.2 indicated a hydroxylation site at C(21). The configuration at this C-atom has been defined
by a NOE experiment where HC(21) showed NOE effects with both Me(29) and
Me(30), indicating an a-orientation of the OH group. This assumption was supported by the multiplicity of HC(21) signal, which appeared as a broad triplet at 3.52 (J ¼ 3.0).
The ESI-MS of compound 8 displayed a quasi-molecular-ion peak at m/z 475.5 ([Mþ H]þ), indicating the molecular formula C
30H50O4with six degrees of unsatura-tion. Fragment-ion peaks at m/z 457.3 ([M H2Oþ H]þ), 439.2 ([M 2 H2Oþ H]þ) were also detected. The1H-NMR spectrum showed signals of six geminal H-atoms bound to an oxygenated function (two as double doublets at d(H) 3.72 and 3.62, and three as doublets at d(H) 3.82, 3.76, and 3.54, and one at d(H) 3.30, obscured by the solvent). In the upfield region, six Me singlets were evident at d(H) 1.26, 0.96, 2 0.92, 0.76, and 0.68. The13C-NMR (Table) showed two O-bearing CH signals at d(C) 78.2 and 74.4, and those of two O-bearing CH2groups at d(C) 67.9 and 64.5. The presence of two sp2 quaternary C-atoms (d(C) 139.9 and 129.8) indicated a C(13)¼C(18) bond. Analysis of COSY, NOESY, HSQC, and HMBC spectra allowed us to determine the structure of 3b,16b,23,28-tetrahydroxyolean-13,18-ene for this compound. This pro-posal was confirmed by the HMBC of Me(27) with the downfield olefinic C(13) and correlations of HC(16) and CH2(28) with the upfield olefinic C(18). The 1H,1
COSY experiment showed correlations between HC(16) (d(H) 3.72) and CH2(15) (d(H) 1.98 and 1.35); and HC(3) (d(H) 3.62) and CH2(2) (d(H) 1.63). The latter correlated with CH2(1) (d(H) 1.71 and 1.05). Finally, H-atoms CH2(12) (d(H) 2.72 and 1.89) correlated with CH2(11) (d(H) 1.27) that correlated with HC(9) (d(H) 1.59). The correlations in the HMBC experiment between CH2(23) (d(H) 3.54 and 3.30) with the C-atom with a signal at d(C) 49.0, assigned to C(5) and bonded to H-atom with the signal at d(H) 1.18, and between this H-atom with the C-atom with the signal at d(C) 74.4 evidenced a OH group at the C(23) position. In the NOESY experiment, the HC(16) showed NOE effects with Me(27) and H-atom HaC(22), strongly suggesting a b-orientation for the OH group; moreover, one of the H-atoms Me(28) (d(H) 3.82) showed a NOE with Me(26) (d(H) 0.92), HC(12) (d(H) 2.72) showed a NOE effect with CH2(19) (d(H) 2.35), and this latter gave NOEs with Me(29) and Me(30) (d(H) 0.96 and 0.76, resp.). The structure of this triterpene was reported some time ago by Shibata et al. [15] as a hydrogenation product of saikogenin A.
The ESI-MS of compound 9 showed a quasi-molecular-ion peak at m/z 473.2 ([Mþ H]þ), suggesting the molecular formula C
30H48O4and seven degrees of unsaturation. Fragment-ion peaks at m/z 455.2 ([M H2Oþ H]þ), as well as an absorption band at 3376 cm1 in the IR spectrum indicated the presence of OH groups. The retro-DielsAlder fragmentation-ion peak, characteristic of the oleane skeleton, in particular the positively charged D/E ring-fragment peak at m/z 249.1 (42%) and the less abundant, positively charged A/B ring-fragment peak at m/z 225.4 (6%) evidenced an olean-12-ene derivative. The IR spectrum also showed absorption bands at 1118, 1090, and 1045 cm1, consistent with the presence of HOC groups, and a band at 1722 cm1 for the presence of a CO group. The13C-NMR (Table) spectrum showed 30 signals, which with the DEPT experiment, revealed the presence of six Me groups (d(C) 31.8, 27.9, 24.8, 18.5, 17.8, and 16.4), an olefinic C-atom (d(C) 124.4), an O-bearing CH group (d(C) 68.3), and two O-bearing CH2 groups (d(C) 69.5 and 68.5). The 1H-NMR spectrum indicated six tertiary Me groups (d(H) 1.28, 1.11, 1.07, 0.98, and 2 0.93), an O-bearing CH group as double doublet (d(H) 4.28), two O-bearing CH2groups (two doublets at d(H) 3.87 and 3.63), and it exhibited two signals (d(H) 3.29 and 3.38), obscured by the solvent, and a trisubstituted olefinic H-atom signal at d(H) 5.29 attributed to HC(12). Analysis of COSY, NOESY, HSQC, and HMBC spectra allowed us to deduce the structure of 16b,23,28-trihydroxyolean-12-en-3-one for this compound. The 1H,1H-COSY experiment showed correlations between: HC(16) (d(H) 4.28) and CH2(15) (d(H) 1.88 and 1.43); CH2(2) (d(H) 2.48 and 2.40) and the CH2(1) (d(H) 1.88 and 1.50); and the olefinic H-atom HC(12) and CH2(11) (d(H) 1.98). The correlations, in the HMBC experiment, between the H-atoms with the signals at d(H) 3.63 and 3.38 with the CH group at d(C) 48.7, assigned to C(5) and bonded to H-atom with a signal at d(H) 1.99, and between this latter H-atom with the O-bearing CH (d(C) 68.5) localized an OH group at C(23). Additionally, the HMBCs between the H-atoms resonating at d(H) 3.63 and 3.38; and Me(26) (d(H) 0.93) with the CO (d(C) 219.2); evidencing the C(3)¼O group and identifying the Me(23) group; and those between the H-atom with a signal at d(H) 2.22, bonded to CH, resonating at d(C) 45.6 and assigned to C(18), with the O-bearing CH with a signal at d(C) 68.3 indicated an OH group at C(28). The configuration of the OH group at C(16)
4.28) and Me(27) (d(H) 1.28) and HC(19), indicating a b-orientation for the OH group.
The ESI-MS of compound 10 showed a quasi-molecular-ion peak at m/z 489.2 ([Mþ H]þ), suggesting the molecular formula C
30H48O5 and seven degrees of unsaturation. Fragment-ion peaks at m/z 471.3 ([M H2Oþ H]þ), 453.1 ([M 2 H2Oþ H]þ), and a retro-DielsAlder fragmentation-ion peak at m/z 267.2 (20%) were characteristic for an olean-12-ene derivative. The IR spectrum showed absorption bands at 3350, 1118, 1090, and 1045 cm1, consistent with the presence of a OH and COC groups, and a band of a CO group at 1724 cm1. The 13C-NMR spectrum (Table) displayed 30 signals, which, with the DEPT experiment, revealed the presence of six Me groups (d(C) 30.1, 27.7, 18.3, 18.0, 17.7, and 16.3), an olefinic C-atom (d(C) 124.8), two O-bearing CH groups (d(C) 74.4 and 68.9), and two O-bearing CH2groups (d(C) 68.4 and 68.3). The1H-NMR spectrum revealed the presence of six tertiary Me (d(H) 0.89, 0.93, 0.96, 1.07, 1.10, and 1.28), two O-bearing CH groups (double doublets at d(H) 4.20 and 3.55), two O-bearing CH2groups (doublets at d(H) 3.60 and 3.78), and it exhibited two signals obscured by the solvent (d(H) 3.30 and 3.32), and one trisubstituted olefinic H-atom signal at d(H) 5.31 attributed to HC(12). Analysis of COSY, NOESY, HSQC, and HMBC spectra allowed us to elucidate the structure of 16b,21b,23,28-tetrahydroxyolean-12-en-3-one for this compound. Many of the corre-lations observed were similar to those of compound 9, indicating the same substitution pattern in rings A, B, C, and D. Another OH group was assigned to C(21) on the basis of the HMBCs between Me(29) and Me(30) (d(H) 0.96 and 0.89, resp.) with the O-bearing CH group resonating at d(C) 74.4, and between HC(21) (d(H) 3.55) and the C-atoms resonating at d(C) 48.6 (C(19)), 45.2 (C(17)), 37.7 (C(20)), 30.1 (C(29)), and 18.0 (C(30)). The configuration of the OH group at C(21) has been determined by a NOESY experiment, where the H-atom with a signal at d(H) 3.55 showed NOE with Me(29) and HC(16) revealing a b-orientation for the OH group.
The ESI-MS of compound 11 exhibited a quasi-molecular-ion peak at m/z 505.3 ([Mþ H]þ), suggesting the molecular formula C
30H48O6 and seven degrees of unsaturation. Fragment-ion peaks at m/z 487.2 ([M H2Oþ H]þ), 469.4 ([M 2 H2Oþ H]þ), and retro-DielsAlder fragmentation-ion peak at m/z 283.1 (20%) evidenced an olean-12-ene derivative. The IR spectrum showed absorption bands at 3330, 1128, 1080, and 1050 cm1, consistent with the presence of OH and HOC groups, and a band of a CO group at 1723 cm1. The1H- and13C-NMR spectra were consistent with an olean-12-ene skeleton. The13C-NMR spectrum (Table) displayed 30 signals, which, with the DEPT experiment, revealed the presence of six Me groups (d(C) 30.6, 28.1, 19.2, 18.5, 17.8, and 16.3), an olefinic C-atom (d(C) 125.6), three O-bearing CH groups (d(C) 78.7, 74.4, and 69.8), and two O-bearing CH2groups (d(C) 69.4 and 59.6). The1H-NMR spectrum revealed the presence of six tertiary Me (d(H) 1.30, 1.10, 1.08, 1.00, 0.93, and 0.92), three O-bearing CH groups (one as double doublet at d(H) 4.63) and two as doublets at d(H) 3.98 and 3.52), two O-bearing CH2groups (four doublets at d(H) 3.92, 3.56 and 3.62, 3.36), and one trisubstituted olefinic H-atom (d(H) 5.40) assigned as HC(12). Analysis of COSY, NOESY, HSQC, and HMBC spectra allowed us to determine the structure of 16b,21b,22a,23,28-pentahydroxyolean-12-en-3-one for this compound. Many of the correlations observed were similar to those of compound 10. The other OH group was assigned to C(22) on the basis of COSY that showed
correlation between HC(21) and HC(22) (d(H) 3.52 and 3.98, resp.), and the large coupling constant of 11.0 Hz indicated their 1,2-trans relation. In the HMBC spectrum, correlations between Me(30) (d(H) 0.92) and Me(29) (d(H) 1.00) with the O-bearing HC(21) (d(C) 78.7); and between HC(21) and the C-atoms resonating at d(C) 74.4 (C(22)), 47.5 (C(17)), 30.6 (C(29)), and 19.2 (C(30)) were observed. Furthermore, the O-bearing HC(22) correlated with C-atoms, resonating at d(C) 69.8, 59.6, and 43.3, that were assigned to the C(16), C(28), and C(18), respectively. The configuration of the OH groups was determined by a NOESY experiment, where the HC(21) showed NOE with Me(29) and HC(16), indicating a b-orientation for the OH group, and
HC(22) showed NOE effects with the Me(30) and HC(18), revealing an
a-orientation for the OH group.
The ESI-MS of compound 12 showed a quasi-molecular-ion peak at m/z 475.1 ([Mþ H]þ), suggesting the molecular formula C
30H50O4and six degrees of unsatura-tion. Fragment-ion peaks at m/z 457.3 ([M H2Oþ H]þ) and 439.1 ([M 2 H2Oþ H]þ) were also observed. The13C-NMR spectrum (Table) showed 30 C-atom signals, identified on the basis of a DEPT experiment as those for five Me, twelve CH2, seven CH groups, and six quaternary C-atoms. The 1H-NMR data, together with those derived from an HSQC experiment, evidenced the presence of two olefinic H-atoms (d(H) 4.72 and 4.60), bonded to the C-atom resonating at d(C) 111.1, four H-atoms (two doublets at d(H) 4.10 and 3.44, correlated to the C-atom resonating at d(C) 62.4, and one as doublet at d(H) 3.52, and one at d(H) 3.29 obscured by solvent, correlated to the C-atom resonating at d(C) 67.7), and, finally, two H-atoms (double doublets at d(H) 3.73 and 3.58), assigned to the C-atoms resonating at d(C) 80.0 and 74.2, respectively. In the upfield region, five Me singlets at d(H) 1.69, 1.11, 1.03, 0.90, and 0.68, correlated, in the HSQC, with the C-atom resonances at d(C) 19.9, 17.7, 16.8, 17.5, and 13.0, respectively, were identified. The 1H,1H-COSY experiment showed the following correlations: HC(3) (d(H) 3.58) with CH2(2) (d(H) 1.66 – 1.60), which showed a cross-peak with CH2(1) (d(H) 1.68 – 1.63 and 0.95 – 0.89); HC(16) (d(H) 3.73) with CH2(15) (d(H) 1.82, 1.45 – 1.40); and HC(19) (multiplet at d(H) 2.50 – 2.45) with HC(18) (d(H) 1.58 – 1.50) and CH2(21) (d(H) 2.05 – 2.02, 1.48 – 1.41). The HMBC experiment furnished useful data to solve the structure. In fact, the sp2-C-atom C(29) correlated with Me(30) (d(H) 1.69) and the HC(19) (d(H) 2.50 – 2.45). The H-atoms of Me(30) were also correlated with the C-H-atoms resonating at d(C) 151.6 (C(20)) and 49.6 (C(19)), while HC(19) also displayed correlations with C-atoms, at resonating d(C) 46.2, 33.9, and 19.9, assigned to C(17), C(22), and C(30), respectively. Moreover HC(3) (d(H) 3.58) and CH2(23) (d(H) 3.52, 3.29), in the same HMBC experiment, gave cross-peaks with C-atoms resonating at d(C) 49.4, 43.9, and 13.0, assigned to C(5), C(4), and C(24), respectively. Finally, HC(16) (d(H) 3.73) and CH2(28) (d(H) 4.10 and 3.44) gave cross-peaks with C(22) and those, resonating at d(C) 49.8 and 46.2, assigned to C(18) and C(17), respectively. These data were in accordance with a lupane triterpene structure. The relative configurations of the stereogenic C-atoms were determined by a NOESY experiment. The H-atoms of Me(27) showed NOE with the HC(16), indicating a b-configuration of the OH group. These data allowed us to determine the structure of 3b,16b,23,28-tetrahydroxylup-20(29)-ene for this compound.
We thank the Associazione Italiana per la Promozione delle Ricerche su Ambiente e Salute umana (AIPRAS-Onlus) for financial support, and CIMCF (Centro Interdipartimentale di Metodologie Chimico-Fisiche) of University of Naples Federico II.
Experimental Part
General. Optical rotations: in MeOH or CH2Cl2; Perkin-Elmer 141 polarimeter. IR Spectra: Jasco
FT/IR430 instrument. HPLC: Shimadzu LC-10AD by using a refractive-index detector Shimadzu RID-10A. Semiprep. HPLC: RP-18 (LiChrospher 10 mm, 250 10 mm i.d.; Merck) column with a flow rate of 1.2 ml min1. Column chromatography (CC): Merck Kieselgel 60 (SiO
2; 230 – 400 mash). Prep. TLC:
silica gel (UV254 precoated) plates with 0.5- and 1.0-mm thickness (Merck).1H- and13C-NMR Spectra:
Varian INOVA-500 FT NMR spectrometer (1H at 499.710 and13C at 125.663 MHz), in CDCl
3or CD3OD
solns., at 258, d in ppm, J in Hz. Proton-detected heteronuclear correlations were measured using a gradient heteronuclear single-quantum coherence (HSQC), optimized for1J(H,C)¼ 140 Hz, and a
gradient heteronuclear multiple bond coherence (HMBC), optimized fornJ(H,C)¼ 8 Hz.
Plant Material. Gymnema sylvestre was purchased from Mother Herbs Ltd. (13 Street, Madhu Vihar, Patpadganj, Delhi – 110 092, India, e-mail: info@motherherbs.com) and identified by Prof. Antonino Pollio of the Dipartimento delle Scienze Biologiche of the University of Naples. A sample specimen (HERBNAWY 124) has been deposited with the herbarium of the University Federico II.
Extraction and Isolation. Dried and finely powdered aerial parts of G. sylvestre (7.0 kg) were sliced and extracted with H2O (25 l for 24 h) and successively with CH2Cl2(20 l for 96 h). The org. extract was
filtered and evaporated in vacuo to remove CH2Cl2. The resulting extract (350 g) was fractionated into
acidic and neutral fractions with aq. 2n NaOH soln. The neutral soln., washed with H2O and concentrated
in vacuo (175 g), was subjected to CC (SiO2; with petroleum ether (PE), CH2Cl2, AcOEt, Me2CO,
MeOH, and H2O).
The fraction eluted with CH2Cl2(25.0 g) was fractionated by CC (SiO2; CH2Cl2/MeOH 100 : 0 to
0 : 100).
The fractions eluted with CH2Cl2(592 mg) were purified by flash CC (SiO2, and then the fractions
eluted with CH2Cl2/AcOEt 17 : 3 (37 mg) were further purified by prep. TLC (CH2Cl2/Me2CO 17 : 3) to
give triterpene 1 (5 mg).
The fractions eluted with CH2Cl2/MeOH 19 : 1 (913 mg) were purified by CC (SiO2), and the
fractions eluted with CH2Cl2/MeOH 19 : 1 (62 mg), were further purified by HPLC (RP-18; MeOH/
MeCN/H2O 2 : 7 : 1) to yield triterpenes 3, 4, 9, and 13 (2, 2, 15, and 2 mg, resp.).
The fractions eluted with CH2Cl2/MeOH 9 : 1 (96 mg) were purified by HPLC (RP-18; MeOH/
MeCN/H2O 3 : 4 : 3) to yield triterpene 6 (36 mg).
The fractions eluted with CH2Cl2/MeOH 9 : 1 (2.28 g) were purified by CC (SiO2), and then fractions
eluted with CH2Cl2/MeOH 4 : 1 (291 mg) were purified by HPLC (RP-18; MeOH/MeCN/H2O 2 : 2 : 1) to
give triterpenes 8 and 12 (3 and 15 mg, resp.).
The fractions eluted with CH2Cl2/MeOH 1 : 9 (6.38 g) were purified by flash CC (SiO2), and the
fractions eluted with Me2CO (611 mg) were purified by HPLC (RP-18 SepPak; MeOH/MeCN/H2O
1 : 2 : 2) to give Frs. 1 – 6. Fr. 4 (60 mg) was purified HPLC (RP-18; MeOH/MeCN/H2O 2 : 3 : 5) to give
triterpene 11 (6 mg). Fr. 5 (127 mg) was purified by HPLC (RP-18; MeOH/MeCN/H2O 2 : 5 : 3) to give
triterpenes 2, 5, and 7 (43, 12, and 10 mg, resp.). Fr. 6 (25 mg) was purified by HPLC (RP-18; MeOH/ MeCN/H2O 2 : 5 : 3) to give triterpene 10 (22 mg).
3b,16b,21b,23-Tetrahydroxyolean-12-ene (¼ (3b,16b,21b)-Olean-12-ene-3,16,21,23-tetrol; 5). Amor-phous powder. [a]25
D¼ þ 20.5 (c ¼ 0.27, MeOH). IR (film): 3347, 1130, 1080, 1035.1H-NMR (CD3OD):
5.28 (br. s, HC(12)); 4.10 (dd, J ¼ 11.7, 3.9, HC(16)); 3.61 (dd, J ¼ 11.0, 4.4, HC(3)); 3.53 (d, J ¼ 11.3, HaC(23)); 3.52 (dd, J ¼ 10.8, 4.2, HC(21)); 3.31 (obscured by solvent, HbC(23)); 2.20 (dd, J ¼ 14.1,
3.8, HC(18)); 2.10 (br. d, J ¼ 13.1, CH2(19)); 1.98 – 1.93 (m, CH2(2), CH2(11)); 1.70 – 1.60 (m, CH2(22));
1.71 – 1.56 (m, 1 H of CH2(7)); 1.69 – 1.59 (m, 1 H of CH2(15)); 1.69 – 1.61 (m, 1 H of CH2(1)); 1.65 – 1.59
(m, HC(5)); 1.50 – 1.42 (m, CH2(6)); 1.33 – 1.27 (m, 1 H of CH2(7)); 1.27 – 1.21 (m, 1 H of CH2(15)); 1.23
0.95 (s, Me(29)); 0.87 (s, Me(30)); 0.81 (s, Me(28)); 0.71 (s, Me(24)).13C-NMR (CD
3OD): see the Table.
ESI-MS: 475.2 ([Mþ H]þ). HR-ESI-MS: 475.3769 ([Mþ H]þ, C
30H51Oþ4; calc. 475.3787).
3b,16b,21a,23,28-Pentahydroxyolean-12-ene (¼ (3b,16b,21a)-Olean-12-ene-3,16,21,23,28-pentol; 7). Amorphous powder. [a]25
D¼ þ 32.3 (c ¼ 0.23, MeOH). IR (film): 3330, 1115, 1095, 1035. 1H-NMR (CD3OD): 5.29 (br. s, HC(12)); 4.57 (dd, J ¼ 11.7, 3.9, HC(16)); 3.74 (d, J ¼ 10.8, HaC(28)); 3.62 (dd, J¼ 11.0, 4.4, HC(3)); 3.54 (d, J ¼ 11.3, HaC(23)); 3.52 (br. t, J ¼ 3.0, HC(21)); 3.32 (obscured by solvent, HbC(28), HbC(23)); 2.30 (dd, J ¼ 14.1, 3.8, HC(18)); 2.17 (br. d, J ¼ 13.1, CH2(19)); 2.16 – 2.10 (m, 1 H of CH2(22)); 1.99 – 1.94 (m, CH2(11)); 1.82 – 1.74 (m, 1 H of CH2(15)); 1.79 – 1.70 (m, 1 H of CH2(22)); 1.73 – 1.67 (m, CH2(2)); 1.71 – 1.66 (m, 1 H of CH2(7)); 1.69 – 1.62 (m, 1 H of CH2(1)); 1.66 – 1.61 (m, HC(9)); 1.52 – 1.43 (m, 1 H of CH2(6)); 1.41 – 1.34 (m, 1 H of CH2(7)); 1.41 – 1.33 (m, 1 H of CH2(6)); 1.33 – 1.25 (m, 1 H of CH2(15)); 1.26 (s, Me(27)); 1.22 – 1.15 (m, HC(5)); 1.05 – 1.00 (m, 1 H of
CH2(1)); 1.03 (s, Me(26)); 1.01 (s, Me(25)); 0.95 (s, Me(29)); 0.92 (s, Me(30)); 0.71 (s, Me(24)).13C-NMR
(CD3OD): see the Table. ESI-MS: 491.4 ([Mþ H]þ). HR-ESI-MS: 491.3728 ([Mþ H]þ, C30H51Oþ5; calc.
491.3736).
3b,16b,23,28-Tetrahydroxyolean-13(18)-ene (¼ (3b,16b)-Olean-13(18)-ene-3,16,23,28-tetrol ; 8). Amorphous powder. [a]25
D¼ 1.2 (c ¼ 0.19, MeOH). IR (film): 3334, 1112, 1087, 1034. 1H-NMR (CD3OD): 3.82 (d, J¼ 11.4, HaC(28)); 3.76 (d, J ¼ 11.4, HbC(28)); 3.72 (dd, J ¼ 12.8, 4.1, HC(16)); 3.62 (dd, J¼ 10.9, 4.9, HC(3)); 3.54 (d, J ¼ 11.7, HaC(23)); 3.30 (obscured by solvent, HbC(23)); 2.72 (br. d, J¼ 15.0, 1 H of CH2(12)); 2.35 (d, J¼ 14.1, 1 H of CH2(19)); 2.33 – 2.28 (m, CH2(21)); 1.98 (br. t, J¼ 12.8, HC(15)); 1.89 (br. t, J ¼ 15.7, 1 H of CH2(12)); 1.73 – 1.66 (m, 1 H of CH2(1)); 1.68 – 1.63 (m, HC(19)); 1.65 – 1.59 (m, CH2(2)); 1.65 – 1.56 (m, CH2(21)); 1.62 – 1.54 (m, HC(9)); 1.51 – 1.45 (m, 1 H of CH2(6)); 1.38 – 1.33 (m, 1 H of CH2(6)); 1.37 – 1.30 (m, 1 H of CH2(15)); 1.27 (m, CH2(11)); 1.26 (s, Me(27)); 1.24 (m, CH2(7)); 1.20 – 1.15 (m, HC(5)); 1.06 – 1.01 (m, 1 H of CH2(1)); 0.96 (s, Me(29)); 0.92
(s, Me(25), Me(26)); 0.76 (s, Me(30)); 0.68 (s, Me(24)).13C-NMR (CD
3OD): see the Table. ESI-MS:
475.5 ([Mþ H]þ). HR-ESI-MS: 475.3745 ([Mþ H]þ, C
30H51Oþ4; calc. 475.3787).
16b,23,28-Trihydroxyolean-12-en-3-one (9). Amorphous powder. [a]25
D¼ þ 25.5 (c ¼ 0.21, MeOH). IR (film): 3376, 1722, 1118, 1090, 1045.1H-NMR (CD 3OD): 5.29 (br. s, HC(12)); 4.28 (dd, J ¼ 12.0, 4.8, HC(16)); 3.87 (d, J ¼ 10.0, HaC(28)); 3.63 (d, J ¼ 10.0, HaC(23)); 3.38 (obscured by solvent, HbC(23)); 3.29 (oscured by solvent, HbC(28)); 2.49 – 2.40 (m, CH2(2)); 2.24 – 2.19 (m, HC(18)); 2.18 – 2.12 (m, 1 H of CH2(22)); 2.02 – 1.75 (m, CH2(19)); 1.99 – 1.96 (m, HC(5)); 1.99 – 1.94 (m, CH2(11)); 1.89 – 1.83 (m, 1 H of CH2(1)); 1.89 – 1.85 (m, 1 H of CH2(15)); 1.76 – 1.71 (m, 1 H of CH2(7)); 1.77 – 1.72 (m, HC(9)); 1.52 – 1.45 (m, CH2(6)); 1.51 – 1.48 (m, 1 H of CH2(1)); 1.45 – 1.39 (m, 1 H of CH2(22)); 1.45 – 1.38 (m, 1 H of CH2(21)); 1.44 – 1.40 (m, 1 H of CH2(15)); 1.43 – 1.39 (m, 1 H of CH2(7));
1.28 (s, Me(27)); 1.25 – 1.20 (m, 1 H of CH2(21)); 1.11 (s, Me(26)); 1.07 (s, Me(25)); 0.98 (s, Me(29)); 0.93
(s, Me(24), Me(30)).13C-NMR (CD
3OD): see the Table. ESI-MS: 473.2 ([Mþ H]þ). HR-ESI-MS:
473.3620 ([Mþ H]þ, C
30H49Oþ4; calc. 473.3631).
16b,21b,23,28-Tetrahydroxyolean-12-en-3-one (10). Amorphous powder. [a]25
D¼ þ 26.5 (c ¼ 0.22,
MeOH). IR (film): 3350, 1724, 1118, 1090, 1045.1H-NMR (CD
3OD): 5.31 (br. s, HC(12)); 4.20 (dd, J ¼
11.7, 4.8, HC(16)); 3.78 (d, J ¼ 10.8, HaC(28)); 3.60 (d, J ¼ 10.8, HaC(23)); 3.55 (dd, J ¼ 11.7, 3.90,
HC(21)); 3.32 (oscured by solvent, HbC(28)); 3.30 (oscured by solvent, HbC(23)); 2.33 – 2.27 (m,
HC(18)); 2.30 – 1.50 (m, CH2(22)); 2.00 (m, CH2(11)); 1.98 (br. d, J¼ 12.3, 1 H of CH2(15)); 1.92 – 1.86
(m, 1 H of CH2(1)); 1.89 – 1.75 (m, 1 H of CH2(7)); 1.88 – 1.82 (m, 1 H of CH2(2)); 1.88 – 1.80 (m,
HC(9)); 1.83 – 1.78 (m, 1 H of CH2(6)); 1.82 – 1.77 (m, 1 H of CH2(19)); 1.75 – 1.70 (m, HC(5)); 1.52 –
1.47 (m, 1 H of CH2(1)); 1.50 – 1.45 (m, 1 H of CH2(7)); 1.40 – 1.35 (m, 1 H of CH2(2)); 1.40 – 1.31 (m, 1 H
of CH2(6)); 1.33 – 1.28 (m, 1 H of CH2(19)); 1.28 (br. d, J¼ 12.3, 1 H of CH2(15)); 1.28 (s, Me(27)); 1.10 (s,
Me(26)); 1.07 (s, Me(25)); 0.96 (s, Me(29)); 0.93 (s, Me(24)); 0.89 (s, Me(30)).13C-NMR (CD
3OD): see
the Table. ESI-MS: 489.2 ([Mþ H]þ). HR-ESI-MS: 489.3563 ([Mþ H]þ, C
30H49Oþ5; calc. 489.3580).
16b,21b,22a,23,28-Pentahydroxyolean-12-en-3-one (11). Amorphous powder. [a]25
D¼ þ 23.1 (c ¼ 0.27, MeOH). IR (film): 3330, 1723, 1128, 1080, 1050.1H-NMR (CD 3OD): 5.39 (t, J¼ 3.4, HC(12)); 4.63 (dd, J¼ 11.6, 5.2, HC(16)); 3.98 (d, J ¼ 11.0, HC(22)); 3.92 (d, J ¼ 10.9, HaC(28)); 3.62 (d, J ¼ 10.8, HaC(23)); 3.56 (d, J ¼ 10.9, HbC(28)); 3.52 (d, J ¼ 11.0, HC(21)); 3.36 (d, J ¼ 10.8, HbC(23)); 2.68 (dd, J¼ 14.2, 4.3, HC(18)); 2.50, 2.38 (2m, CH2(2)); 2.05 (m, 1 H of CH2(11)); 1.98 (m, HC(9)); 1.96 (m, 1 H of CH2(11)); 1.92 (m, 1 H of CH2(19)); 1.90, 1.50 (2m, CH2(1)); 1.77 (dd, J¼ 11.5, 6.1, HC(5));
1.71 (m, 1 H of CH2(7)); 1.68 (br. d, J¼ 12.3, 1 H of CH2(15)); 1.48 (m, CH2(6)); 1.36 (br. d, J¼ 12.7, 1 H
of CH2(7)); 1.30 (s, Me(27)); 1.26 (m, 1 H, Me(15)); 1.20 (dd, J¼ 14.0, 4.5, 1 H of CH2(19)); 1.10 (s,
Me(26)); 1.08 (s, Me(25)); 1.00 (s, Me(29)); 0.93 (s, Me(24)); 0.92 (s, Me(30)).13C-NMR (CD
3OD): see
the Table. ESI-MS: 505.3 ([Mþ H]þ). HR-ESI-MS: 505.3500 ([Mþ H]þ, C
30H49Oþ6; calc. 505.3529).
3b,16b,23,28-Tetrahydroxylup-20(29)-ene (¼ (3b,16b)-Lup-20(29)-ene-3,16,23,28-tetrol; 12). Amor-phous powder. [a]25
D¼ þ 10.5 (c ¼ 0.23, MeOH). IR (film): 3350, 1556, 1090, 1045.1H-NMR (CD3OD):
4.72 (br. s, HC(29)); 4.60 (br. s, HC(29)); 4.10 (d, J ¼ 11.5, HaC(28)); 3.73 (dd, J ¼ 10.5, 5.0, HC(16)); 3.58 (dd, J ¼ 11.0, 5.0, HC(3)); 3.52 (d, J ¼ 11.5, HaC(23)); 3.44 (d, J ¼ 11.5, HbC(28)); 3.29 (overlapped, HbC(23)); 2.50 – 2.45 (m, HC(19)); 2.37 (br. dd, J ¼ 12.5, 7.9, 1 H of CH2(22)); 2.05 – 2.02 (m, 1 H of CH2(21)); 1.82 (br. t, J¼ 12.2, 1 H of CH2(15)); 1.69 (s, Me(30)); 1.71 – 1.63 (m, 1 H of CH2(12)); 1.68 – 1.63 (m, 1 H of CH2(1)); 1.66 – 1.60 (m, CH2(2)); 1.59 – 1.40 (m, CH2(7)); 1.58 – 1.50 (m, HC(18)); 1.48 – 1.41 (m, 1 H of CH2(21), CH2(6)); 1.46 – 1.23 (m, CH2(11)); 1.45 – 1.40 (m, 1 H of CH2(15)); 1.38 – 1.31 (m, HC(9)); 1.22 – 1.17 (m, 1 H of CH2(22)); 1.11 (s, Me(26)); 1.12 – 1.07 (m, HC(5)); 1.05 – 1.01 (m, 1 H of CH2(12)); 1.03 (s, Me(27)); 0.95 – 0.89 (m, 1 H of CH2(1)); 0.90 (s, Me(25)); 0.68 (s, Me(24)).13C-NMR (CD
3OD): see the Table. ESI-MS: 475.1 ([Mþ H]þ). HR-ESI-MS:
475.3765 ([Mþ H]þ, C
30H51Oþ4; calc. 475.3787).
REFERENCES
[1] L. D. Kapoor, CRC Handbook of Ayurvedic Medicinal Plants, CRC Press, Boca Raton, FL, 1990, p. 200.
[2] W. Ye, X. Liu, Q. Zhang, C.-T. Che, S. Zhao, J. Nat. Prod. 2001, 64, 232.
[3] K. Yoshikawa, K. Amimoto, S. Arihara, K. Matsuura, Tetrahedron Lett. 1989, 30, 1103. [4] P. Kanetkar, R. Singhal, M. Kamat, J. Clin. Biochem. Nutr. 2007, 41, 77.
[5] N. Shigematsu, R. Asano, M. Shimosaka, M. Okazaki, Biol. Pharm. Bull. 2001, 24, 713. [6] M. S. Granich, B. P. Halpern, T. Eisner, J. Insect Physiol. 1974, 20, 435.
[7] N. Komalavalli, M. V. Rao, Plant Cell Tissue Org. Cult. 2000, 61, 97. [8] E. Porchezhian, R. M. Dobriyal, Pharmazie 2003, 58, 5.
[9] P. Rasoanaivo, G. Multari, E. Federici, C. Galeffi, Phytochemistry 1995, 39, 251. [10] K. Yoshikawa, A. Mizutani, Y. Kan, S. Arihara, Chem. Pharm. Bull. 1997, 45, 62. [11] K. Yoshikawa, H. Taninaka, Y. Kan, S. Arihara, Chem. Pharm. Bull. 1994, 42, 2023.
[12] A. Garcia-Granados, P. E. Lopez, E. Melguizo, A. Parra, Y. Simeo, J. Org. Chem. 2007, 72, 3500. [13] K. Yoshikawa, K. Amimoto, S. Arihara, K. Matsuura, Chem. Pharm. Bull. 1989, 37, 852.
[14] H. Neukirch, M. DAmbrosio, S. Sosa, G. Altinier, R. Della Loggia, A. Guerriero, Chem. Biodiversity 2005, 2, 657.
[15] S. Shibata, I. Kitagawa, H. Fujimoto, Chem. Pharm. Bull. 1966, 14, 1023.
